Differentiated cells are capable of being reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with embryonic stem (ES) cells. Additionally, studies have shown that mouse embryonic or adult fibroblasts can be induced to become pluripotent cells by using retroviral vectors to induce expression of four factors: Oct3/4, Sox2, c-Myc and Klf3. These induced cells are termed induced pluripotent stem cells (iPS). The iPS cells exhibit the morphology and growth properties of ES cells and express ES cell maker genes. Upon injection into nude mice these cells form tumors, severely limiting their potential use as therapeutic agents. (See, e.g., Takahashi, K. and Yamanaka, S., Induction of Pluripotent Stem Cells from mouse Embryonic and Adult Fibroblast Cultures by Defined Factors, Cell, 126:663-676 (2006)).
MicroRNAs (miRNAs) are single-stranded RNA molecules, typically between 21 and 23 nucleotides in length. They are endogenously occurring, untranslated RNA molecules involved in regulation of gene expression. miRNAs are also important regulators of development and differentiation. (See, e.g., Suh, et al., Human embryonic stem cells express a unique set of microRNAs, Developmental biology, 270:488-498 (2004). miRNAs function to regulate gene expression through targeting mRNAs for cleavage or translation repression. (See, e.g., Bartel, D. P., MicroRNAs: Genomics, Biogenesis, Mechanism, and Function Cell 116:281-297 (2004).) At present, nearly all of the identified miRNAs are conserved in closely related animals, such as humans and mouse. (See, e.g., Lagos-Quintana et al., New microRNAs from mouse and human RNA 9:175-179 (2003); Lim et al., Vertebrate microRNA genes Science 299:1540 (2003)).
One microRNA cluster, designated the miR-290 cluster, constitutes over 70% of the entire miRNA population in mouse ES cells (Marson, A. et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells Cell 134:521-533 (2008)). Expression of the miR-290 cluster is rapidly down-regulated upon ES cell differentiation (See, e.g., Houbaviy, H. B., Murray, M. F. & Sharp, P. A. Embryonic stem cell-specific MicroRNAs Dev Cell 5:351-358 (2003)). A subset of the miR-290 cluster, called the embryonic stem cell cycle (ESCC) regulating miRNAs, enhances the unique stem cell cycle and includes miR-291-3p, miR-294, and miR-295, as well as the human homologues hsa-mir-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302d, hsa-miR-371-5p, hsa-miR-372, hsa-miR-373. (See, e.g., Wang, Y. et al. Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation Nat Genet 40:1478-1483 (2008)). This subset includes miR-291-3p, miR-294, and miR-295 and their homologues.
Removal of genes required for maturation of all miRNAs has shown that miRNAs play essential roles in the proliferation and differentiation of Embryonic Stem Cells (ESCs)(Wang, Y. et al., Nat Genet 39:380-5 (2007); Kanellopoulou, C. et al. Genes Dev 19:489-501 (2005); Murchison, E. P. et al., Proc Natl Acad Sci USA 102:12135-40 (2005)). For example, the loss of the RNA binding protein DGCR8, which is required for the production of all canonical miRNAs, results in a cell cycle defect and an inability to silence the self-renewal program of ESCs when they are placed in differentiation-inducing conditions (Wang, Y. et al., Nat Genet 39:380-5 (2007). The introduction of individual members of a family of miRNAs, the ESCC miRNAs, into Dgcr8−/−ESCs can rescue the cell cycle defect (Wang, Y. et al., Nat Genet, 40:1478-1483 (2008)). We have discovered that these same miRNAs are able to enhance the de-differentiation of somatic cells to iPS cells and also have identified another large family of miRNAs, the let-7 family, which performs the opposite role to the ESCC family. When introduced into Dgcr8−/−ESCs, let-7 silences self-renewal by suppressing many of the same downstream targets that are indirectly activated by the ESCC family. Indeed, co-introduction of the ESCC miRNAs inhibits the capacity of let-7 to silence self-renewal, and suppression of the let-7 family in somatic cells promotes de-differentiation.